Quantitation of antigen in tissue by immunofluorescence image analysis

John M. Basgen, Thomas E Nevins, Alfred F. Michael

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13 Scopus citations


A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/μg) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.

Original languageEnglish (US)
Pages (from-to)77-83
Number of pages7
JournalJournal of Immunological Methods
Issue number1
StatePublished - Nov 13 1989

Bibliographical note

Funding Information:
We thank Christine Alonzi, Tom Groppoli, Sue Kupcho-Sandburg and Karen Clugge for excellent technical assistance, Paul Walker for the preparation and quantitation of radiolabeled IgG. We are also grateful to the staff of the Biomedical Image Processing Laboratory for help and assistance with the image analysis system and Dr. Ruth 0sterby for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (Al10704) and the Viking Children's Fund.


  • (Kidney)
  • Basement membrane
  • Fluorescence
  • Image processing
  • Immunohistochemistry


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